J. Natn.Sci.Foundation Sri Lanka 2005 33(3): 169-186
REVIEW
LICHENS: A CHEMICALLY IMPORTANT BIOTA
VERANJA
KARUNARATNE",
KARUNANANDA
KATHIRGAMANATHAR and VINITHA M. THADHANI
BOMBUWELA,
SELVALUXMY
Department of Chemistry, University of Peradeniya, Peradeniya.
(Accepted: 03 October 2005)
Abstract: Lichens produce a wide variety of secondary
metabolites which assist them in the colonization of terrestrial
h a b i t a t s . S u c h compounds a r e i m p o r t a n t i n t h e
chcmotaxonomy of lichens. Furthermore, lichen substances
exhibit antiherbivore, antimicrobial and antineoplastic
activities. This review highlights the different types of lichen
substances and their activities, uses and t h e possible
ecological role.
Lichens a r e symbiotic organisms of fungi
(mycobiont) and algae (photobiont) comprising
about 17,000 species. Lichens are unique because
they look and behave quite differently from their
component organisms. They are classified and
named according to their fungal partner, with
many thousands of species of lichens sharing a
much smaller number of photobiont species.
Lichens show a world-wide distribution commonly
growing on rocks and poorly developed soils such
as those of arid lands and boreal-arctic regions or
as epiphytes on trees and shrubs.
Primary metabolites of lichens, which are
intracellular, are proteins, amino acids, polyols,
carotenoids, polysaccharides and vitamins.
Lichens produce a wide array of secondary
metabolites (extracellular). There are over 700
lichen substances reported to date1and many are
restricted to t h e lichenised s t a t e . Broadly
speaking, there are three major types of lichen
substances based on their biosynthetic pathway^.^
(a) Depsides, depsidones and dibenzofurans
formed by the acetate-malonate pathway. The
most important of these are the esters and
the oxidative coupling products of simple
phenolic units related to orcinol and p-orcinol.
Most depsides and depsidones are colourless
compounds which occur in the medulla of the
lichen. However, usnic acids, yellow cortical
*
Corresponding author
compounds formed by the oxidative coupling
of methylphloroacetophenone units are found
in the cortex of many lichen species (Figure
1). Anthraquinones, xanthones and
chromones, are all pigmented compounds
which occur in the cortex, which are also
produced by the acetate-malonate pathway by
intramolecular condensation of long folded
polyketide units rather than the coupling of
phenolic units (Figure 2).
(b) The shikimic acid biosynthetic pathway
produces two major groups of pigmented
compounds,which occur in the cortex: pulvinic
acid derivatives and terphenyl quinones
(Figure 3). Although most pulvinic acid
derivatives lack nitrogen, they a r e
biosynthesized through phenyl alanine.
Nitrogen is strongly limiting to metabolic
activities in most lichens, and nitrogen rich
metabolites such as alkaloids are unknown
among lichen substances.
(c) Terpenes and steroids are produced by the
mevalonic acid pathway. These include
compounds unique to lichens and many that
occur in higher plants as well (Figure 4).
Chemotaxonomy of lichens
The application of chemical discriminators to
lichen taxonomy is widely accepted. The secondary
metabolites in about 5000 species (33 % of all
known species) have been studied and the data
are used in the routine identification of lichens
than in any other group of organism^.^^^
Cortical chemistry: It has now been recognized by
many lichenologsts that some cortical substances
Veranja Karunaratne et al.
Alectorialic acid
(Benzyl depside)
Lecanoric acid
(para-Depside)
Sekikaic acid
(meta-Depside)
Gyrophoric acid
(Tri depside)
Micareic acid
(Diphenyl ether)
Didymic acid
(Dibenzofuran)
Usnic acid
Virensic acid
(Depsidone)
Picrolichenic acid
(Depsone)
Contortin
(Biphenyl)
Figure 1: Structures of typical acetyl-polymalonyl lichen products derived from two or more
polyketide chains.
Lichens: a chemically important biota
Aliphatic acids
H02C,
H
,C02H
(CH2)11CH3
H3C
(+)-Protolichesterinicacid
H
-
"
Roccellic acid
Mononuclear phenolic compounds
Orsellinic acid
Siphulin (Chromone)
Haemoventosin (Naphthaquinone)
POrsellinic acid
Lichexanthone (Xanthone)
Averythrin (Anthraquinone)
Figure 2: Structures of some acetyl-polymalonyl lichen products derived from a single polyketide
chain.
Veranja Karunaratne et al.
172
Pulvinic acid derivatives
Pulvinic acid
Calycin
Terphenyl quinones
Polyporic acid
Indolyl-3-acetic acid
Thelephoric acid
Ambewelamide A, 1 R = (CH,),CH,
Ambewelamide B, 2 R = (CH,),CH,
Figure 3: Structures of some lichen products derived from the shikimic acid pathway.
Terpenoids
(-)-p-Thujone(Monoterpenoid)
(-)-Sandaracopimaricacid (Diterpenoid)
Fukinanolide (Sesquiterpenoid)
Hopan-Ga,7a,22-triol (Triterpenoid)
Lichens: a chemically important biota
Ergosterol (Steroid)
y-Carotene (Carotenoid)
Figure 4: Structures of typical lichen products derived from the mevalonic acid pathway.
are correlated with higher taxonomic ranks. For
example, vulpinic acid in Letharia (at the genus
level) or anthraquinones such as parietin in
Teloschistaceae (at the family l e ~ e l ) . ~
Medullary chemistry
Variations in medullary constituents are used
mainly as discriminators at the species level. Most
morphologically defined species have a constant
chemistry, regardless of their geographical
location, substrate or ecology. Within a complex
of morphologically similar species, three common
patterns of chemical variation can be discerned:
(a) replacement compounds, (b) chemosyndromic
variation and (c) accessory type compound^.^
With replacement compounds, cogeneric
chemotypes show simple replacement of one
substance by another. Morphologically these
lichen
populations
are
sometimes
indistinguishable, but possess well-defined and
constant variations in chemical compositions. For
example, Psuedovernia furfuraceae has three
chemical races. One race contains olivetoric acid
and is found in Northern Europe, a second is found
in Southern Europe and Northern Africa and
contains physodic acid and third containing
lecanoric acid is found i n North America.
Biogenetically the first two compounds are closely
related because one can be derived from the other
by one single step. But lecanoric acid is not
biosynthetically related to these two compounds.
I t is therefore now recognized t h a t when
biogenetic
demarcations
complement
biogeographic variations, such taxa should be
recognized as separate species. Thus, the North
American taxon is named as P. c o n s ~ c i a n s . ~
However, it has been suggested that the best way
to recognize whether chemical variation is under
genetic control or not is through sympatric
chemical races which maintain their chemical
integrity even when growing side by side."
Chemosyndrome refers to a group of
biosynthetically related lichen substances and the
major metabolite is accompanied by several minor
constituents which are biosynthetically relatede4
The compounds which are major constituents in
one species may occur as minor constituents in
related taxa and vice versa. These chemotypes can
also be considered as sibling species5, where
reproductive isolation of a species often leads to
ecological differentiation. This is a common result
of evolution in animals and vascular plants.
The occurrence of accessory compounds
may tend to complicate the above arguments.
These compounds are found at certain times in
species, usually i n addition to normal
constituents, a n d have relation to the
morphological or distributional variation. Thus,
these
compounds
a r e not
accorded
chemotaxonomic significance. Such compounds
Veranja Karunaratne et al.
often occur as accessory compounds in more than
one species and often vary in quantity.
The following spot tests and microchemical
methods will be used to garner taxonomical details
of lichen specimens:
(a) Chemical (colour) tests conducted on lichen
thalli
The common spot test reagents not only
indicate where particular compounds are located
in sectioned thalli, but may also indicate the
chemical nature of the substance.
Reagents for thalline spot tests:
K = 10 % aqueous KOH solution
a. Turns yellow to red with most o-hydroxy
aromatic aldehydes
b. T u r n s b r i g h t red to deep p u r p l e w i t h
anthraquinone pigments
C = saturated aqueous Ca(OCl), or common bleach
NaOC1 solution
a. Turns red with m-dihydroxy phenols, except
for those substituted between the hydroxy
groups with a -CHO or -CO,H.
b. Turns green with dihydroxy dibenzofurans.
KC = 10 % aqueous KOH solution followed by
saturated aqueous Ca(OCl), or common
bleach (NaOC1) solution.
a. Turns yellow with usnic acid.
b. Turns blue with dihydroxy dibenzofurans.
c. Turns red with depsides and depsidones
which, undergo rapid hydrolysis to yield mdihydroxy phenolic moiety.
PD = 5 % alcoholic p-phenylenediamine solution
a. Turns yellow, orange or red with aromatic
aldehydes.
(b) Microchemical methods
by the position relative to the standards. Data are
then sorted to find all compounds with the same
R,classes. Of these possibilities, those with similar
spot characteristics (colour, fluorescence etc.,) are
compared chromatographically w i t h t h e
unknown. Additional solvent systems are used for
compounds that do not separate well in the initial
analysis. Two-dimensional TLC methods are used
to evaluate structurally similar compounds.
Before spraying the dried plates are examined in
day light for pigments and for fluorescence or
quenching u n d e r s h o r t (254 n m ) a n d long
wavelength (365 nm) ultraviolet (UV) light.
Subsequently the plates are sprayed with 10 %
sulfuric acid and heated a t 110" C on a hot plate
for 10 minutes to develop the spots. The following
solvent systems are used:
Solvent A = Toluene: dioxane: acetic acid
(180: 45: 5)
Solvent B = Hexane: methyl tert. butyl ether:
formic acid (140: 72: 18)
Solvent C = Toluene: acetic acid (170: 30)
Additional solvents:
Solvent E = Cyclohexane: ethyl acetate (75: 25)
Solvent G = Toluene: ethyl acetate: formic acid
(139: 83: 8)
(ii) High performance liquid chromatography
(HPLC): Samples are dissolved in methanol and
eluted through the appropriate partition column
by using a suitable solvent or solvent system
u n d e r high pressure. The substances t h a t
separate are detected in a UV detector. The
retention time R, and peak intensity are recorded
by a chart recorder. HPLC is also used to measure
either absolute or relative concentrations of lichen
compounds. Gradient elution methods are used.
The RI value of an unknown peak is calculated as
follows:
RI = R, of Peak - R, of benzoic acid xlOO
R, of Solorinic acid
silica gel plates a n d t h r e e solvent systems
(designated A, B & C) and two internal controls
RI values are very stable during the
(atranorin and norstictic acid) are ~ s e d . All
~,~
Rf, ~
lifetime of a column. Substances which are eluted
values are compared. An acetone extract of the
a t t h e s a m e time often belong to different
lichen is spotted on the plate and subsequently
substance classes and can be distinguished by
eluted in each solvent system. For each solvent
their UV spectra.
system, a spot is assigned to a R,class determined
(i) Standardized TLC methodology: Commercial
Lichens: a chemically important biota
(iii) Gas Chromatography and Mass Spectrometry:
Xanthones, anthraquinones, dibenzofurans,
terpenes a n d pulvinic acid which lack
thermolabile ester groups can be studied by gas
chromatography coupled with mass spectrometry
(GCMS). Xanthones in lichens were studied by
injecting a lichen extract directly into a mass
spe~trometer.~
More recently, the main terpenoid
components of t h e lichens of t h e family
Pyxinaceae have been studied by GCMS."
Ecological role of lichen substances
A significant number of lichen substances are not
present in non-lichenised fungi. Many genera of
lichens also do not produce any of t h e
characterized products. Thus the presence of these
compounds are not required to maintain the
symbiotic state.
Lichen substances are found deposited in
either the cortex or the medulla as crystals or on
the outer surface of hyphae. The commonest
cortical compounds are usnic acid and atranorin.
Anthraquinones, pulvinic acid derivatives and
xanthones also occur in the cortex to a lesser
degree. All these compounds with the exception
of atranorin are pigmented. The majority of lichen
substances a r e deposited i n t h e medulla.
Concentrations of lichen substances are variable
and typically it is around 1% but values of 5-6 %
a r e not uncommon. Once formed, lichen
substances are very stable. In lichens, there is no
correlation between growth r a t e s a n d
concentration of lichen substances, although high
concentrations are found at actively growing lobe
or branch tips as well as soralia and apothecial
margins. There appear to be several roles of lichen
substances which determine t h e relative
ecological success of individual lichen species and
the structure and diversity of natural lichen
communities: (a) light-screening compounds, (b)
chemical withering compounds, (c) biologically
active compounds, (d) anti-herbivore defense
compounds and (e) Allergenic compounds:l
(a) Light-screening compounds
Many cortical lichen substances show variation
in concentration along light gradients. Evidence
suggests that these compounds act as lightscreens, regulating the light intensity reaching
the algal layer in the upper cortex. Usnic acid, a
yellow cortical pigment is the most widespread
lichen substance found in thousands of lichens.
Lichen populations having usnic acid which are
exposed to the sun are yellow, while the shade
populations are grey-green, indicating a lower
concentration of the depside. Cladonia subtenuis
and Parmeliopsis ambigua show concentrations
of usnic acid varying along concentration
gradients.ll
Anthraquinones such as parietin ( a
reddish cortical pigment) appear to act like usnic
acid acting as a light filter. This compound is
characteristic of species within Teloschistaceae.
These species occur in exposed habitats. In
extreme desert environments, species of
Caloplaca may be the only saxicolous lichens
present, whileXanthoria and Caloplaca dominate
highly exposed coastal areas. Pigmented
xanthones in the outer cortex of many crustose
lichens may also play a role in regulating the light
intensity reaching t h e algal layer. These
xanthones which are yellow or brown in colour
occur in many species of Lecanora, Pertusaria,
Melanaria, Lecidea and Buellia in habitats with
high light intensities. Unlike fungal xanthones,
lichen xanthones contain one or more chlorinated
substituents, suggesting that the availability of
chloride in the environment may affect the
production of these compound^.^
(b) Chemical withering compounds
Lichen substances such as phenolic compounds
with carbonyl functional groups play an important
role in the withering of rock due to their ability to
complex metal ions.12 This process leads to soil
formation. Low but significant amounts of these
substances dissolve in water. Solubility is
determined by the nature and the number of polar
groups present. The metal complexation i~
advantageous to many crustose and foliose lichens
growing in close proximity to rock substratez
Since lichen substances commonly occur as
crystals on the outer surfaces of hyphae, it puts
them in direct contact with the substrate where
biochemical withering could aid attachment.
176
Veranja Karunaratne et al.
(c) Biologically active compounds
The biological activity of lichen compounds very
likely gives them a n ecological advantage in
colonization of terrestrial areas. Such compounds
have been used by man as well. Lichens have been
used in medicine by the ancient Chinese and
Egyptians.l"14 The medicinal use of lichens is not
prevalent at present, particularly since the testing
of the atom bomb and the Chernobyl accident in
1986 which have increased the concentrations of
radionuclides (137Cs a n d 40K) to a l a r m i n g
prop~rtions.~%ccording
to recent investigations,
lichens possess a variety of biological activities:
( i ) A n t i m i c r o b i a l a c t i v i t y : Although lichen
substances are used as antibiotics, their activity
is found to be low compared to those isolated from
microorganisms. Furthermore, lichen antibiotics
are not adequately water soluble for therapeutic
use. Depsides, depsidones and usnic acid are
active against Gram-positive microorganism^.^^
Sodium salt of usnic acid was used as a drug in
Russia.17 Antiseptic creams such as "Usno" and
"Evosin" contain usnic acid. Usnic acid acts by
uncoupling phosphorylation and is effective
because animal cells are far less permeable to
usnic acid than the microorganisms.
In S r i Lanka, lichens h a d remained
completely unexplored (both chemically and
taxonomically) until 1996, when a systematic
study to harness their biological and medicinal
v a l u e was i n i t i a t e d a t t h e D e p a r t m e n t of
Chemistry, University of Peradeniya.
Roccella montagnei is a fruticose lichen,
which is pendulous or shrubby. It is sparsely to
moderately branched and has shades of grey,
specially lilac grey. (Figure 5).lDifferent Roccella
lichen species a r e used to prepare complex
mixtures of pigments for the preparation of litmus
paper (pH 4.5 red; pH 8.3 blue). Two compounds
Erythrin
namely, erythrin and methylorsellinate have been
isolated from the dichloromethane extract of
Roccella
montagnei
collected
from
Bamunakotuwa in Kurunegala district.
I n g e n e r a l , e r y t h r i n showed lower
antifungal activity when compared with benomyl
(Benlate- standard funpcide) in the TLC bioassay
technique. Apart from somewhat reasonable
activity shown a g a i n s t C o l l e t o t r i c h u m
gleosporioides, erythrin exhibited moderate
activity against four fungi namely, Colletotrichum
musae, Cladosporium cladosporioides, Curvularia
trifolii, and Monacrosporium ambrosium. These
results were corroborated by a spore germination
a s s a y a g a i n s t C o l l e t o t r i c h u m m u s a e . The
germination was 70.1 %with erythrin at 10 ppm,
while for the untreated control germination was
74.2 %.
Methylorsellinate on the other hand,
showed significantly higher antifungal activity as
compared to erythrin (Table 1).Methylorsellenate,
when
tested
against
Colletotrichum
gleosporioides,
Colletotrichum
musae,
Cladosporium cladosporioides and Curvularia
t r i f o l i i , showed g r e a t e r activity t h a n t h e
commercial fungicide Benlate. Methylorsellinate
showed lesser activity against t h e fungus,
Monacrosporium a m b r o s i u m as compared to
Benlate. Based on the diameter of inhibition
zones, methylorsellinate was significantly more
active than erythrin against C. cladosporioides.
The
antifungal
activity
of
methylorsellinate was also confirmed by a spore
germination assay using Colletotrichum musae.
The
germination
percentage
with
methylorsellinate was 29.9 %while in the control
it was 74 %.
In order to show that methylorsellinate
was not produced as a n artifact in the methanol
Methylorsellenate
Lichens: a chemically important biota
Figure 5: Roccella montagnei lichen growing on the trunk of a coconut tree, Cocos nucifera
Table 1: Antifungal activity of erythrin and methylorsellinate
Inhibition zone diameter 1 mm
Fungus
Erythrin(41)
Methylorsellinate(42)
Benlate
Colletotrichum musae
25
50
Colletotrichum gloeosporioides
20
44
25
Cladosporium cladosporioides
10
50
31
Curvularia trifolii
16
55
25
Monacrosporium ambrosium
10
36
48
extract by the methanolysis of erythrin, the lichen
Roccella montagnei was extracted into ethyl
acetate. Upon examination by TLC and co-TLC it
was evident that this extract also contained both
erythrin and methylorsellinate, proving that the
latter was indeed a natural product isolated from
Roccella montagnei and not an artifact.
is found in large amounts in the lichen (4.9 % of
dry weight) compared with methylorsellinate (0.3
% of dry weight). It is not uncommon that lichen
substances are accumulated in amounts exceeding
20 % of the dry weight of the thallus. These
compounds are considered to impart a protective
role against invading pathogens and herbivores.18
Both erythrin and methylorsellinate are
almost identical in functionality and in general,
phenolic compounds a r e known to exhibit
antifungal properties. In fact the total hydrolysis
of erythrin would yield two molecules of orsellinic
acid and such the latter could be considered as
the biosynthetic precursor of the former. Erythrin
Interestingly, in Roccella montagnei the
storage of hydrolytically labile erythrin in large
amounts (with its relatively low antifungal
activity) leads us to reason that the lichen may
be converting erythrin to the strongly antifungal
methylorsellinate when required.
Veranja Karunaratne et aL.
Figure 6: Usnea species from Ambewela growing on decaying Acacia decurrens
Ambewelamide A, R = (CH2),CH3
Ambewelamide B, R = (CH2),CH3
(ii) Anti tumour and anti mutagenic activity: (-1Usnic
acidlg, protolichesterinic
and
nephrosteranic acids20,physodalic acid20~21,
lichen
g l u c a n ~and
~ ~lichenin derivatives" have shown
anti tumour and anti mutagenic activity. Lichen
polysaccharides, the class of compounds to which
the latter two belong are particularly interesting
due to their anti tumour activity. The mechanism
of action of these lichen polysaccharides are not
completely known, b u t a p p e a r s to be host
mediated: such compounds a r e believed to
g e n e r a t e lymphoid cells, p l a s m a cells,
macrophages i n t h e vicinity of t h e grafted
tumo~r.~~
It was found that extracts of the lichen
Usnea sp., collected from Ambewela, exhibited
potent in vitro cytotoxicity (Figure 6 ) . Bioassay
guided fractionation of the Usnea sp. extracts led
to the isolation of ambewelamides A (1)and B (21,
whose structures have been elucidated via a
combination of single crystal X-ray diffraction and
spectroscopic a n a l y s e ~ . ~ ~ ~ ~ ~
Ambewelamides A (1)and B (2)are new members
of a family of highly modified diketopiperazines
that include fungal metabolites such as a r a n ~ t i n ~ ~ ,
apoaranotinZ7,emethallicinsZ8,exserohiloneZ9and
SCH64847.30There are reports of anti allergenic
28,anti viralz7,EGF receptor binding 30,phytotoxic
29 activity for t h e members of t h e family of
diketopiperazines, but the ambewelamides are the
first to show potent cytotoxicity. Ambewelamides
are the only members of the family having ring Al
Lichens: a chemically important biota
E epoxides and a diketopiperazine epidisulphide
bridge, suggesting that these functionalities might
be necessary for cytotoxicity. The ambewelamides
are the first examples of the diketopyperazine family isolated from a lichen.
(d) Anti-herbivore compounds
herbivores. Since these compounds are bright
yellow in colour, their cortical position indicates
a n example of warning coloration. The medullary
position of pulvinic acid derivatives i n t h e
Stictaceae and two species ofParmelio. suggest a
defensive role against small, nonvisual1y.-oriented
invertebrate herbivores.ll
In vascular plants, several studies have
Both vertebrate and invertebrate herbivores
shown that sesqui- and diterpenoids may act as
consume lichens. The reasons for the low observed
defense restricting h e r b i ~ o r yThe
. ~ ~nutrient rich
levels of herbivory may be due to low nutrient
species of Pseudocyphellaria, Sticta a n d
content of lichens, the difficulties in metabolising
lichens, and the sparse nature of lichens as a
Nephroma show a surprising lack of herbivory
indicating that terpenes may play an important
resource. Lichens comprise a major source of
role in protecting these species with high nitrogen
winter food supply of' caribou and reindeer in
content. l1
boreal a r e a s i n t h e Northern Hemisphere.
Although lichens are a poor source of proteins (2Fumarprotocetraric acid is a b i t t e r
3 %), they are a n easily accessible carbohydrate
source when higher plant foliage is not a ~ a i l a b l e . ~ ~depside occurring in many lichens which appear
to deter herbivory. For example, neither Caldonia
In addition, herbivory on lichens by insects, mites,
arbuscula nor C. rangiferina (both contain
molluscs are i m p ~ r t a n t .Despite
~ ~ . ~ the
~ ~ lichens
~~
fumarprotocetraric acid) are eaten by reindeer,
being a potential food source for herbivores,
while they s r e attracted to C. alpestris which has
feeding on lichens is not common. In temperate
no fumarprotocetraric acid.39Concentrations of
evergreen forests, there is little evidence of
f'umarprotocetraric acid vary greatly between
v e r t e b r a t e or i n v e r t e b r a t e herbivory. T h e
younger and older thallus parts in Cladonia, with
resistance of individual lichens toherbivory may
t h e g r e a t e s t concentration occurring in
be due to a variety of factors. Lichens with
metabolically active growing tips." Species
gelatinous sheaths covering their surface are
containing depsides and depsidones such as
relatively immune to herbiv01-y~~
as seen in some
diffractic acid, physodic and physodalic acids and
species of Collemataceae. Thick cortical structure
salazinic acid which are bitter are avoided by
with heavily agglutinated hyphae may also
Caribou in Canada.41
provide some protection from small invertebrate
herbivores. Inorganic chelate agents present in
P a r i e t i n , a n a n t h r a q u i n o n e lichen
many saxicolous crustose lichens are toxic to
substance appears to have a defensive role against
numerous insects.36 F o u r groups of lichen
herbivore^.^^ Lichen species of Teloschistaceae
substances that have possible defensive functions
contain
relative high contents of ~ a r i e t i n . ~ ~
in lichens are pulvinic acid derivatives, terpenes,
depsides and depsidones and anthraquinones.
(+)-Usnicacid isolated from a n Usnea sp.
i n t h e Ambewela a r e a 4 4showed significant
Several studies support t h e primary
antitermite activity (80 % mortality at 10 mg)
defensive role of pulvinic acid derivatives. Lichens
against Glyptotermes dilatatus, which is a major
containing these compounds have relatively high
live-wood tea-termite a t low elevations. Although
nitrogen content thus making them potentially a
usnic acid is a well-known antimicrobial
valuable food source. Yet, feeding on these taxa
compound16 t h e r e is only one report of t h e
are virtually unknown. Vulpinic acid, the most
insecticidal activity of usnic acid against the
widespread of these compounds is toxic to both
polyphagous herbivorous insect Spodoptera
vertebrates and invertebrate^.^^ The cortical
l i t t o r ~ l i s . 'Currently,
~
the control methods of G.
presence of most of the pulvinic acid derivatives
dilatatus are focused on t h e development of
is unusual since they show no variation along light
r e s i s t a n t clones a n d t h e r e a r e no reported
gradients. B u t t h i s histological position is
chemicals which can be used in controlling the
consistent with their defensive role against
180
Veranja Karunaratne et al.
pest, and as such search for such compounds are
imp~rtant.~"
(e) Allergenic activity
Contact d e r m a t i t i s a m o n g forestry a n d
horticultural workers has been attributed to the
exposure to lichen substances. Among the many
lichen substances responsible for such allergenic
action a r e usnic acid, evernic acid,
fumarprotocetraric acid, stictic acid and atranorin.
A t r a n o r i n a n d stictic acid a r e capable of
photosensitizing human skin as well as being
contact allergen^.^^ Atranorin and other p-orcinol
type depsides absorb UV radiation and this may
account for their photo toxicity.
Chemical ecology of lichens
Sequestering of plant chemicals by insects is a
well-known phenomenon. Specifically, butterflies
of the family Lycaenidae are known to sequester
plant chemicals.47Larvae of Monarch butterflies
(Nymphalidae) a c c u m u l a t e toxic cardiac
glycosides in mill. weeds, which are used by the
a d u l t s to d e t e r predators.48 Although t h e
sequestration of lichen compounds by the moths
of the families Arctiidae and Lithosiinae are
known, butterflies are not known to sequester
lichen compounds.
Many species of invertebrates live on and
among lichens, using them for concealment,
shelter or food. Over half of the orders of insects
have associations with lichens.49
Moths belonging to the family Lepidoptera
have the richest association with li~hens.~lThere
are many examples of moth names reflecting the
resemblance to lichens, such as the "pale lichen
moth" (Crambidia pallida) and the "powdered
lichen moth" (Bruceiapuluerina). A noctuid moth,
Zanclognatha theralis, in Tennessee, USA, was
discovered to both feed on Usnea stigosa and look
remarkably like the lichen.52
The experiments with polyphagous larvae
of the moth Spodoptera littoralis (Noctuidae)
demonstrated pronounced acute toxicity and
feeding deterrency for frequently occurring lichen
compounds such as (+)- and (-)-usnic acids and
vulpinic acid a t concentrations comparable to or
even below those found in many lichens.53
Hesbacher et a1." have shown that the
lichen phenolics such as parietin, atranorin as well
as the hydrolytic cleavage product of atranorin
(methyl-2, 4-dihydroxy-3, 6-dimethylbenzoate)
among other lichen compounds were detected in
11different moth species of the family Arctiidae.
COOMe
OH
Parietin
Methyl-2, 4-dihydroxy-3, 6-dimethylbenzoate
Atranorin
Lichens: a chemically important biota
(-)-Usnic acid
a-collatolic acid
Vulpinic acid
The moth larvae of Lithosiinae feed on
epiphytic algae of trees or on the algal component
of lichens.48A recent survey for the presence of
sequestered lichen compounds i n several
Lithosiinae species in Australia and Germany,
demonstrated t h a t t h e presence of lichen
compounds is variable but wide spread in wildcaught imagines.48
Many snails and slugs (class: Gastropoda)
feed on lichens. In Israel, two species of snails
that eat lichens growing under the surface of lime
stone rocks were discovered to be converting rock
to soil a t a n amazing rate. The snails pass
significant amounts of rock through their digestive
tracts in the process of consuming the lichen. In
addition, the snails were taking the nitrogen from
the lichen and leaving it behind in the new
On the other hand, many lichen species contain
bitter compounds that may discourage feeding by
invertebrate^.^^ Although the ecological role of the
sequestered compounds for the herbivores
remains unknown, it is thought that they may be
utilized for chemical defense against predators or
pathogen^.^^
~ i g & e7: T. nyseus butterfly landing on B. calycinum
Veranja Karunaratne et al.
Three species of lichen-grazing snails,
Baleaperversa, Chondrina clienta and Helicig-ona
lapicida were found to sequester lichen
compounds when feeding on the crustous lichen
species Aspicilia calcarea, Caloplaca atra and
Xanthoria parietina. The lichen compounds such
as (+)-usnicacid or a-collatolic acid were detected
in the feces of these snails, suggesting selective
uptake of lichen compounds by the snails.57
Lycaenidae is t h e largest family of
butterflies in Sri Lanka and consist of 40 genera,
comprising 8 1 species.50Some species of
Lycaenidae live on plants, a few live or, scaleinsects, others feed on the larvae and pupae of
the large red ant Oecophylla smaragdina, while
many are actually tended by ants in their nests.
Some are cannibalistic, devouring larvae or the
pupae of their own hnd.50Fruit-eating forms are
also found in the Lycaenidae.
Talicada nyseus Guerin (red Pierrot)
which belongs to the family Lycaenidae is
distributed from Sri Lanka to Indo-China. The Sri
Lankan population belongs to the nominotypical
race, which flies through out the island below 4000
feet. The butterflies love human settlement being
equally abundant in domestic gardens as it is in
the
It is plentiful all over the island all the
year round and flies slowly near the ground and
settles f r e q ~ e n t l y .The
~ ~ natural food source of
its larvae is Bryophyllum calycinum (Sinhala:
akkapana; Tamil: malai-kalli) (Figure 7).
During our studies of Sri Lankan lichens,
we discovered the sequestration of lichen products
belonging to the crustaceous lichen Leproloma
sipmanianum Kurnmerl & Leukert by t h e
butterfly T. nyseus Guerin (red pierrot)
(Lycaenidae) in Beragala (80" 54' 30" E, 6"45' 30"
N), Uva province, Sri Lanka.58T. nyseus was found
flying close to the extensive lichen thallus,
growing on road side proterozoic rocks of gneiss
and quartz schist which are exposed at road-edges,
and roosting on it periodically (Figure 8).
Pupae of the butterflies were also located
anchored to the thallus surface. T. nyseus were
collected from Beragala between the period from
September 1996 and March 2001.
Out of seven butterfly collections made,
on three occasions, five compounds namely, zeorin
1, psitosterol 2, a long-chain fatty acid ester 3,
atranorin 4, and (+)-usnic acid 5, were present in
wild-caught imagines (Figure 9). The butterf ies
which emerged from the pupae also contained
these compounds.
The presence of these compounds in the
butterfly was confirmed by comparative TLC
studies a n d GC-MS with authentic lichen
substances isolated from t h e lichen L.
sipmanianum.
The butterflies found at the University of
Peradeniya and Kandy contained only psitosterol
Figure 8: T.nyseus butterfly landing on L. sipmanianum lichen
Lichens: a chemically important biota
CH3(CH2)4ICH20CCH2CH2CH2CH3
Me
H
3
CHO
OH
0
0
Figure 9: Structures of lichen compounds present in L. sipmanianum and T. nyseus: 1. Zeorin, 2. BSitosterol, 3. Fatty acid ester, 4. Atranorin, 5. (+)-Usnicacid.
2 (common plant sterol also found in the host plant
B. calycinum) with no trace of other lichen
compounds. It was concluded that the presence
of lichen products of L. sipmanianum in adult T.
nyseus butterflies indicated that its larvae may
feed on the lichen although the natural food source
is B. calycinum.
Out of the seven butterfly collections made
from Beragala, all five compounds, (1) - (5) were
present in adult butterflies on three occasions.
Importantly, on two occasions, the butterflies
which emerged from laboratory-reared pupae also
contained compounds (1) - (5). (+)-Usnicacid (5)
was present only on three occasions and atranorin
(4) four times. T. nyseus collected during all the
field visits contained zeorin (I), 0-sitosterol (2)
and the fatty acid ester (3).
In order to expand the scope of the study
and investigate the presence of lichen products
in T. nyseus in other areas of the island, butterflies
were collected from four other locations: foot hills
of Adam's peak in Central Province (80"30' 50" E,
6" 50' N), Peradeniya University premises (80"35'
30" E, 7" 15' N), from a domestic garden in
Aniewatta, Kandy (80"38' E, 7" 17' 30" N), both in
the Central Province and in Ukgalkalthota (80"
52' 30" E, 6" 39' 30' N) in the Uva Province.
Butterflies were subjected to the same analysis
protocol as described above using both TLC and
GC-MS. Interestingly, the butterflies found at the
University of Peradeniya and Kandy contained
only 0-sitosterol with no trace of other compounds.
However, those collected in Ukgalkalthota
contained atranorin (4) and 0-sitosterol while
butterflies a t the foot-hills of Adam's peak
contained (+)-usnic acid (5) along with 0sitosterol. Examination of the extracts (hexane,
dichloromethane and methanol) of air-dried plant
material (140 g) of BryophylLum calycinum by
TLC and MPLC did not reveal the presence of
lichen compounds (I), (3), (4) and (5). However,
the dichloromethane extract (1.50 g) whose TLC
showed the presence of p-sitosterol upon MPLC
on silica gel (eluent: s t e p gradient from
dichloromethane: hexane (2: 3) to methanol:
dichloromethane (1:79)) yielded fl-sitosterol as a
colourless crystalline solid (0.021 g) confirming
that B. calycinum contains p-sitosterol 2 and it
could be the source of the butterfly.
It may be concluded that the presence of
lichen products found in L. sipmanianum in adult
Veranja Karunaratne et al.
T. nyseus butterfly indicates that its larvae can
feed on the lichen although its natural food source
is B. c a l y c i n u m . In t h e case of T. n y s e u s
individuals contained only P-sitosterol
(Peradeniya and Kandy), the larvae probably used
the natural food source as their only diet. On the
other hand, the presence of atranorin (4) and (+)usnic acid ( 5 ) , compounds which are found only
in lichens, in the butterflies in Ukgalkalthota and
at the foot-hillsof Adam's peak, respectively, lends
credence to the evidence accumulated in Beragala
that the larvae of T. nyseus may indeed sequester
lichen products.
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